mNeonGreen-tagged fusion proteins and nanobodies reveal localization of tropomyosin to patches, cables, and contractile actomyosin rings in live yeast cells
Open accessPreprint — published as 10.1242/jcs.260288 · Paper page
Abstract
Tropomyosins are structurally conserved α-helical coiled-coil dimeric proteins that bind along the length of filamentous actin (F-actin) in fungi and animals. Tropomyosins play essential roles in the stability of actin filaments in non-muscle cells and are essential for calcium regulation of myosin II contractility in the muscle. Despite the crucial role of tropomyosin in actin cytoskeletal regulation, in vivo investigations of tropomyosin are limited, mainly due to the suboptimal live cell imaging tools currently available in many organisms. Here, we report mNeon-Green (mNG) tagged tropomyosin, with native promoter and linker length configuration, that clearly reports tropomyosin localization and dynamics in Schizosaccharomyces pombe (Cdc8), Schizosaccharomyces japonicus (Cdc8), and Saccharomyces cerevisiae (Tpm1 and Tpm2), in vivo and in isolated S. pombe cell division apparatuses. We extended this approach to also visualize the mammalian TPM2 isoform. Finally, we generated a camelid-nanobody against S. pombe Cdc8, which mimics the localization of mNG-Cdc8 in vivo without significantly influencing cell growth and dynamics of actin cytoskeleton. Using these tools, we report the presence of tropomyosin in previously unappreciated patch-like structures in fission and budding yeasts, show flow of tropomyosin (F-actin) cables to the cytokinetic actomyosin ring, and identify rearrangements of the actin cytoskeleton during mating. These powerful tools and strategies will aid better analyses of tropomyosin and actin cables in vivo .
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Cites
Highly accurate protein structure prediction with AlphaFold
Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., Tunyasuvunakool, K., Bates, R., Žídek, A., Potapenko, A., et al. (2021) · Nature · doi:10.1038/s41586-021-03819-2 · Cited by 43,764 · see citing works
A Modular Design for the Clathrin- and Actin-Mediated Endocytosis Machinery
Kaksonen, M., Toret, C. P., & Drubin, D. G. (2005) · Cell · doi:10.1016/j.cell.2005.09.024 · Cited by 726 · cited 1× · see citing works
“Furthermore, an extensive body of work has been done on the interplay between actin patches, actin cables, and endocytosis (Drubin et al., 2005; Kaksonen et al., 2005; Kaksonen et al., 2006).”
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- Authors
- Hatano, T., Lim, T. C., Billault-Chaumartin, I., Dhar, A., Gu, Y., Massam-Wu, T., Adishesha, S., Springall, L., Sivashanmugam, L., Scott, W., Mishima, M., Martin, S. G., Oliferenko, S., Palani, S., & Balasubramanian, M. K.
- Affiliations
- University of Warwick (GB)
- University of Lausanne (CH)
- Indian Institute of Science Bangalore (IN)
- King's College London (GB)
- Corresponding author
- Hatano, Tomoyuki
- Palani, Saravanan
- Balasubramanian, Mohan K.
- ORCID
- Venue
- Journal of Cell Science
- Publisher
- openRxiv
- Published
- 2022-05-20
- Volume
- 135
- Issue
- 18
- ISSN
- 0021-9533 (ISSN-L)
- PMC
- PMC9592052
- OpenAlex
- W4296807180
- Cited by
- 19
- References
- 2
- Citations by year
- 2026: 3 · 2025: 7 · 2024: 4 · 2023: 3 · 2022: 2
- FWCI
- 2.13
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- cc-by
- Locations
- Journal of Cell Science (publishedVersion, cc-by)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- SERVAL (Université de Lausanne) (publishedVersion, cc-by)
- Warwick Research Archive Portal (University of Warwick) (acceptedVersion)
- Figshare (submittedVersion, cc-by)
- IRIS (submittedVersion, cc-by)
- Research Portal (King's College London) (submittedVersion, other-oa)
- Archive ouverte UNIGE (University of Geneva) (submittedVersion, cc-by)
- The Francis Crick Institute (cc-by)
- Funders
- National Science Foundation
- Indian Institute of Science
- Wellcome Trust (101885/C/13/Z, 220790/Z/20/Z, WT101885MA, 203276/Z/16/Z)
- University of Warwick
- Department of Biotechnology, Ministry of Science and Technology, India
- Department of Science and Technology, Ministry of Science and Technology, India
- Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (176396, 220790)
- The Wellcome Trust DBT India Alliance (IA/I/21/1/505633)
- Directorate for Biological Sciences
- Biotechnology and Biological Sciences Research Council (BB/S003789/1, BB/T000481/1)
- Science and Engineering Research Board (SRG/2021/001600)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Microtubule and mitosis dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular Mechanics and Interactions
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
- Keywords
- tropomyosin, nanobodies, actin cytoskeleton, Schizosaccharomyces pombe, Schizosaccharomyces japonicus, Saccharomyces cerevisiae, live-cell imaging, myosin II contractility, fluorescent probes
- MeSH terms
- Actins, Actomyosin, Animals, Fluorescent Dyes, Mammals, Actin Cytoskeleton, Saccharomyces cerevisiae, Schizosaccharomyces, Tropomyosin, Cell Cycle Proteins, Protein Isoforms, Schizosaccharomyces pombe Proteins, Cytokinesis, Single-Domain Antibodies
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